Dilated Virchow-Robin spaces are a marker for arterial disease in multiple sclerosis. EBioMedicine BACKGROUND:Virchow-Robin spaces (VRS) have been associated with neurodegeneration and neuroinflammation. However, it remains uncertain to what degree non-dilated or dilated VRS reflect specific features of neuroinflammatory pathology. Thus, we aimed at investigating the clinical relevance of VRS as imaging biomarker in multiple sclerosis (MS) and to correlate VRS to their histopathologic signature. METHODS:In a cohort study comprising 142 MS patients and 30 control subjects, we assessed the association of non-dilated and dilated VRS to clinical and magnetic resonance imaging (MRI) outcomes. Findings were corroborated in a validation cohort comprising 63 MS patients. Brain blocks from 6 MS patients and 3 non-MS controls were histopathologically processed to correlate VRS to their tissue substrate. FINDINGS:In our actively treated clinical cohort, the count of dilated centrum semiovale VRS was associated with increased T1 and T2 lesion volumes. There was no systematic spatial colocalization of dilated VRS with MS lesions. At tissue level, VRS mostly corresponded to arteries and were not associated with MS pathological hallmarks. Interestingly, in our ex vivo cohort comprising mostly progressive MS patients, dilated VRS in MS were associated with signs of small vessel disease. INTERPRETATION:Contrary to prior beliefs, these observations suggest that VRS in MS do not associate with an accumulation of immune cells. But instead, these findings indicate vascular pathology as a driver and/or consequence of neuroinflammatory pathology for this imaging feature. FUNDING:NIH, Swedish Society for Medical Research, Swiss National Science Foundation and University of Zurich. 10.1016/j.ebiom.2023.104631